The Ran GTPase is required for nuclear assembly, nuclear transport, spindle assembly, and mitotic regulation. The nucleotide exchange factor for Ran, RCC1, is a chromatin-associated protein. RanGAP1 is the activating protein for the Ran. Ran-GTP nucleotide hydrolysis also requires a family of Ran-GTP binding proteins, which act as RanGAP1 accessory factors. This family includes RanBP1 and RanBP2. Vertebrate RanGAP1 is conjugated to a small ubiquitin-like protein, SUMO-1. This modification promotes association of RanGAP1 with the interphase nuclear pore complex (NPC) through binding to the nucleoporin RanBP2. During mitosis, RanGAP1 is concentrated at kinetochores in a microtubule- (MT) and SUMO-1-dependent fashion. RanBP2 is also abundantly found on kinetochores in mitosis. Interestingly, plant RanGAP1 is targeted to the NPC during interphase and to the cell plate during mitosis, using mechanisms that are completely independent of the SUMO pathway. The distribution of Ran?s regulators has been widely hypothesized to modulate local concentrations of Ran-GTP within cells, spatially directing processes in which Ran has been implicated. We have been examining the mechanisms through which key Ran regulators are localized within mitotic metazoan cells and the functional consequences to cells when such distribution patterns are disrupted. Using RNA interference (RNAi) to deplete cellular RanBP2, we have shown that RanBP2 and RanGAP1 are targeted as a single complex that is both regulated by and essential for stable kinetochore-MT association in mitotic spindles. We have also documented that Crm1, a Ran-GTP-binding nuclear export receptor, localizes to kinetochores. Moreover, Crm1 ternary complex assembly is essential for Ran-GTP-dependent kinetochore recruitment of the RanGAP1/RanBP2 complex. Inhibition of Crm1 using the drug Leptomycin B (LMB) causes release of RanGAP1/RanBP2 from kinetochores and the formation of spindles in which continuous MT bundles span the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibers. These findings demonstrate that proper localization of RanGAP1/RanBP2 is essential for definition of kinetochore fibers and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and chromosome segregation in somatic cells. The spindle assembly checkpoint monitors spindle formation and prevents the onset of the metaphase-anaphase transition until chromosomes are correctly attached and aligned on the metaphase plate. In previous experiments, we documented that the spindle assembly checkpoint can be regulated through Ran-GTP in Xenopus egg extracts. In yeast and vertebrate cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the NPCs during interphase, and we have examined the relationship of these proteins to the Ran pathway in budding yeast. We found that deletion of yeast MAD1 or MAD2 genes did not grossly affect steady-state nucleocytoplasmic trafficking or Ran localization. However, yeast with conditional mutations in the yeast Ran pathway that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. These observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.